Ebola virus (EboV) is readily transmitted and rapidly fatal, and there is no effective vaccine or drug therapy Recent experiments reveal that the physiological trigger for EboV infection is stepwise proteolytic cleavage ol the envelope glycoprotein (GP) by cathepsin L and cathepsin B, two members of a family of endosoma cysteine proteases. Importantly, well-characterized small molecule inhibitors of cathepsin proteases markedly inhibit EboV infection in vitro (>99%). The goal of this proposal is to develop the potential of cathepsir inhibitors as anti-EboV drugs. Specific Aim #1: Develop pipeline of small molecules that inhibit EboV GP-dependent infection a. Screen compounds in major classes of cathepsin inhibitors to identify lead compounds with anti- EboV activity. Include inhibitors already under development for other disorders. b. Determine if any of the 198 specific inhibitors of EboV-GP infection identified by high-throughput screen of 40,000 small molecules are cathepsin inhibitors or function synergistically with them. c. Determine anti-EboV activity of promising lead compounds in existing animal models. Specific Aim #2: Determine role of cathepsins in EboV infection. a. Use selective inhibitors and knockout-derived cell lines to identify all cathepsins that mediate EboV and Marburg virus infection. b. Identify the cathepsins that mediate infection of host tissues where EboV replication is high. c. Develop BL3-based system to select and analyze EboV variants that are dependent on specific cathepsins and/or are resistant to specific inhibitors. Specific Aim #3: Design, synthesize and test second generation EboV inhibitors as part of drug development plan. SA 1-2 will inform the starting point for an iterative lead optimization protocol to identify the most highly selective cathepsin inhibitors that retain potent and broad anti-EboV activity.